Tobacco-specific nitrosamines are among the most important carcinogens in tobacco products. Extensive analytical studies, carried out in the U.S. and internationally, clearly document the presence in tobacco products of substantial quantities of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN), as well as several related compounds. The carcinogenic activities of NNK and NNN in laboratory animals are well-established. These data, together with considerable biochemical evidence from studies with rodent and human tissues, support the prominent role of tobacco-specific nitrosamines as major causative factors for a number of tobacco-related cancers including lung, oral cavity, esophagus, and pancreas. Metabolism and DNA adduct formation are critical factors in the mechanisms of carcinogenesis by NNK, its major metabolite 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanol (NNAL), and NNN. Our goal is to understand these processes and use this knowledge as a basis for developing practical strategies for prevention of tobacco-induced cancer. Our overall hypothesis is that cancer susceptibility relates to carcinogen dose as well as the balance between carcinogen metabolic activation and detoxification. Our specific aims are: 1. Carry out a comprehensive analysis, using liquid chromatography-electrospray ionization-mass spectrometry, of DNA adduct formation and persistence in rats chronically treated with NNK, NNAL, and NNN, and extend these studies to humans. This research builds on our recent structural characterization of pyridyloxobutyl DNA adducts of NNK and NNAL. 2. Determine the levels and persistence of formaldehyde-derived cross-linked and hydroxymethyl DNA adducts in rats treated with NNK, NNAL, and N-nitrosodimethylamine. We have recently shown, for the first time, that the metabolic activation of N-nitrosomethyl carcinogens leads to formation of these formaldehyde-DNA adducts in vitro. 3. Develop methods to determine the balance of metabolic activation and detoxification of NNK in humans. This is critical to an understanding of NNK metabolism as an indicator of cancer susceptibility. 4. Assess the endogenous formation of NNK in smokers, snuff-dippers, and people who use nicotine replacement therapy. This aim extends our observation that 2'-hydroxylation of nicotine leads directly to the precursor of NNK. 5. Develop mass spectrometry methods to analyze nicotine, cotinine, and total NNAL in human toenails, as a biomarker of tobacco smoke exposure. Better biomarkers of chronic tobacco smoke exposure are needed, particularly for studies of environmental tobacco smoke exposure and cancer. The results of these studies will provide exciting new data on mechanisms of nitrosamine carcinogenesis in laboratory animals and humans, and will lead to practical methods to test the hypothesis that human uptake, metabolic activation and detoxification of tobacco-specific nitrosamines are related to cancer susceptibility in humans.